Synthetic Protease Inhibitor Research Articles

Fourteen immunomodulatory alkaloids and two prenylated phenylpropanoids with dual therapeutic approach for COVID-19: molecular docking and dynamics studies

The pandemic outbreak of COVID-19 caused by the new severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a global health burden. To date, there is no highly effective antiviral therapy to eradicate the virus; as a result, researchers are racing to introduce new potential therapeutic agents. Alternatively, traditional immunity boosters and symptomatic treatment based on natural bioactive compounds are also an option. The 3-chymotrypsin-like protease (3CLpro) crystal structure, the main proteolytic enzyme of SARS-CoV-2, has been unraveled, allowing the development of effective protease inhibitors via in silico and biological studies. In COVID-19 infected patients, the loss of lung function, and mortality are reported to be linked to several inflammatory mediators and cytokines. In this context, the approach of introducing immunomodulatory agents may be considered a dual lifesaving strategy in combination with antiviral drugs. This study aims to provide immunomodulatory natural products exhibiting potential protease inhibitory activities. Selected groups of alkaloids of different classes and two prenylated phenylpropanoids from the Brazilian green propolis were in silico screened for their ability to inhibit COVID-19 3CLpro protease. Results showed that compounds exhibited binding energy scores with values ranging from −6.96 to −3.70 compared to the reference synthetic protease inhibitor O6K with a binding energy score of −7.57. O6K binding energy was found comparable with lead phytochemicals in our study, while their toxicity and drug-likeness criteria are better than that of O6K. The activities of these molecules are mainly ascribed to their ability to form hydrogen bonding with 3CLpro crucial amino acid residues of the catalytic site. In addition, the molecular dynamics simulations further showed that some of these compounds formed stable complexes as evidenced by the occupancy fraction measurements. The study suggested that the major immunomodulators 3β, 20α-diacetamido-5α-pregnane, (20S)-(benzamido)-3β-(N,N-dimethyamino)-pregnane, and baccharin are 3CLpro inhibitors. Biological screenings of these phytochemicals will be valuable to experimentally validate and consolidate the results of this study before a rigid conclusion is reached, which may pave the way for the development of efficient modulatory bioactive compounds with dual bioactions in COVID-19 intervention. Communicated by Ramaswamy H. Sarma

Exploiting the co-crystal ligands shape, features and structure-based approaches for identification of SARS-CoV-2 Mpro inhibitors

SARS-CoV-2 enters the host cell through the ACE2 receptor and replicates its genome using an RNA-Dependent RNA Polymerase (RDRP). The functional RDRP is released from pro-protein pp1ab by the proteolytic activity of Main protease (Mpro) which is encoded within the viral genome. Due to its vital role in proteolysis of viral polyprotein chains, it has become an attractive potential drug target. We employed a hierarchical virtual screening approach to identify small synthetic protease inhibitors. Statistically optimized molecular shape and color-based features (various functional groups) from co-crystal ligands were used to screen different databases through various scoring schemes. Then, the electrostatic complementarity of screened compounds was matched with the most active molecule to further reduce the hit molecules’ size. Finally, five hundred eighty-seven molecules were docked in Mpro catalytic binding site, out of which 29 common best hits were selected based on Glide and FRED scores. Five best-fitting compounds in complex with Mpro were subjected to MD simulations to analyze their structural stability and binding affinities with Mpro using MM/GB(PB)SA models. Modeling results suggest that identified hits can act as the lead compounds for designing better active Mpro inhibitors to enhance the chemical space to combat COVID-19.Communicated by Ramaswamy H. Sarma

Structure and activity of human TMPRSS2 protease implicated in SARS-CoV-2 activation.

Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-CoV-2. Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) protein, enabling virus-host membrane fusion and infection of the airways. We present here a recombinant production strategy for enzymatically active TMPRSS2 and characterization of its matured proteolytic activity, as well as its 1.95 Å X-ray cocrystal structure with the synthetic protease inhibitor nafamostat. Our study provides a structural basis for the potent but nonspecific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that explain specificity. TMPRSS2 cleaved SARS-CoV-2 S protein at multiple sites, including the canonical S1/S2 cleavage site. We ranked the potency of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.4 nM to 120 µM and determined inhibitor mechanisms of action, providing the groundwork for drug development efforts to selectively inhibit TMPRSS2.

Therapeutic Strategies for Disseminated Intravascular Coagulation Associated with Aortic Aneurysm.

Aortic aneurysms are sometimes associated with enhanced-fibrinolytic-type disseminated intravascular coagulation (DIC). In enhanced-fibrinolytic-type DIC, both coagulation and fibrinolysis are markedly activated. Typical cases show decreased platelet counts and fibrinogen levels, increased concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer, and increased FDP/D-dimer ratios. Thrombin-antithrombin complex or prothrombin fragment 1 + 2, as markers of coagulation activation, and plasmin-α2 plasmin inhibitor complex, a marker of fibrinolytic activation, are all markedly increased. Prolongation of prothrombin time (PT) is not so obvious, and the activated partial thromboplastin time (APTT) is rather shortened in some cases. As a result, DIC can be neither diagnosed nor excluded based on PT and APTT alone. Many of the factors involved in coagulation and fibrinolysis activation are serine proteases. Treatment of enhanced-fibrinolytic-type DIC requires consideration of how to control the function of these serine proteases. The cornerstone of DIC treatment is treatment of the underlying pathology. However, in some cases surgery is either not possible or exacerbates the DIC associated with aortic aneurysm. In such cases, pharmacotherapy becomes even more important. Unfractionated heparin, other heparins, synthetic protease inhibitors, recombinant thrombomodulin, and direct oral anticoagulants (DOACs) are agents that inhibit serine proteases, and all are effective against DIC. Inhibition of activated coagulation factors by anticoagulants is key to the treatment of DIC. Among them, DOACs can be taken orally and is useful for outpatient treatment. Combination therapy of heparin and nafamostat allows fine-adjustment of anticoagulant and antifibrinolytic effects. While warfarin is an anticoagulant, this agent is ineffective in the treatment of DIC because it inhibits the production of coagulation factors as substrates without inhibiting activated coagulation factors. In addition, monotherapy using tranexamic acid in cases of enhanced-fibrinolytic-type DIC may induce fatal thrombosis. If tranexamic acid is needed for DIC, combination with anticoagulant therapy is of critical importance.

Molecular docking and simulation studies of synthetic protease inhibitors against COVID-19: a computational study

COVID-19 is the most recent threat to global health. Many people preferred treatment in case of infection instead of vaccination. The inhibition of viral replication is a good strategy for the treatment of COVID-19 infection. 3CLpro and PLpro are two important viral proteases responsible for proteolysis, infection, and replication of the virus. Therefore, targeting of these two enzymes is an attractive way to deal with COVID-19. The aim of this study was to screen some synthetic protease inhibitors to determine an appropriate hit molecule against COVID-19 using molecular docking and molecular dynamic simulations. The strategy depends on docking existing synthetic compounds mostly HIV protease inhibitors against two COVID-19 proteases to identify promising drugs for the treatment of COVID-19. We used protein data bank to obtain the X-ray crystal structure of the most important COVID-19 proteases 3CL pro (PDB ID: 6M2N) and PL pro (PDB ID: 6WX4). In this conceptual context, an attempt has been made to suggest an in silico computational relationship between 50 synthetic protease inhibitors and COVID-19 proteases. Out of 50 screened compounds, the best docking scores were found for these five protease inhibitors BDBM7021, BDBM698, BDBM694, BDBM93239, BDBM700. A 100-ns MD simulation was carried out to assess the stability of COVID-19 proteases and inhibitors, revealing an average RMSD value of 0.7 and favorable binding free energy (MM-GBSA) for all complexes confirming their potency as powerful binders in the COVID-19 proteases’ binding pocket. Furthermore, the current results must be confirmed using in-vitro and in-vivo antiviral methods. Communicated by Ramaswamy H. Sarma

Proteinase inhibitors in legume herbivore defense: from natural to genetically engineered protectants.

Proteinase inhibitors (PIs) from legumes have the potential for use as protectants in response to pests and pathogens. Legumes have evolved PIs that inhibit digestive proteinases upon herbivory resulting in delayed development, deformities, and reduced fertility of herbivorous insects. Legume PIs (serine proteinase inhibitors and cysteine proteinase inhibitors) have been overexpressed in plants to confer plant protection against herbivores. Recently, the co-expression of multiple PIs in transgenic plants enhanced host defense over single PI expression, i.e., in an additive fashion. Therefore, a synthetic PI could conceivably be designed using different inhibitory domains that may provide multifunctional protection. Little attention has yet given to expanding PI gene repertoires to improve PI efficacy for targeting multiple proteinases. Also, PIs have been shown to play an important role in response to abiotic stresses. Previously published papers have presented several aspects of strategic deployment of PIs in transgenic plants, which is the focus of this review by providing a comprehensive update of the recent progress of using PIs in transgenic plants. We also emphasize broadening the potential usefulness of PIs and their future direction in research, which will likely result in a more potent defense against herbivores.

Management of Disseminated Intravascular Coagulation in Emergency: A Review

Disseminated intravascular coagulation (DIC) is life threatening disease it’s often associated with sepsis, which require ICU management. In individuals with sepsis, the risk of DIC is especially high, DIC affects 30 to 50 percent of these individuals. Also it affects only about ten percent of patients with solid tumors, trauma, or obstetric emergencies. Hematological malignancies, aneurysms, and liver diseases can also cause the disease, the treatment for DIC focuses on resolving the underlying problem that led to this condition in the first place. As a result, antibiotics for severe sepsis, delivery in the case of placental abruption, and exploratory surgical intervention in the case of trauma are the pillars of DIC treatment, Patients with active bleeding or a high risk of bleeding, as well as those who require an invasive surgery, should consider platelet and plasma transfusions. Other anti-coagulant drugs can also be used. Prothrombin complex concentrates should only be administered in an emergency, due to their possible dangers. Recombinant human soluble thrombomodulin rhTM it was developed and licensed for clinical usage in Japan in 2008, and it’s one of the novel treatments for DIC. Anti-Xa agents, Synthetic protease inhibitors, and antithrombin are another options for treatment. In this article we will be making overview of the disease, it’s etiology an what’s the current management options.

Nafamostat mesilate, a nuclear factor kappa B inhibitor, enhances the antitumor action of radiotherapy on gallbladder cancer cells.

Nuclear factor kappa B (NF-κB) is a transcriptional factor that can be activated by radiotherapy and chemotherapy. The synthetic protease inhibitor nafamostat mesilate (NM) inhibits NF-κB activity and exerts antitumor actions in various types of cancer. In the present study, we hypothesized that NM might enhance the antitumor action of radiotherapy on gallbladder cancer (GBC) cells by inhibiting radiation-induced NF-κB activity. Thus, we investigated the correlation between radiotherapy and NF-κB activity in GBC cells. We assessed the in vitro effects of radiotherapy with or without NM on NF-κB activity, apoptosis of GBC cells (NOZ and OCUG-1), induction of apoptotic cascade, cell cycle progression, and viability of GBC cells using four treatment groups: 1) radiation (5 Gy) alone; 2) NM (80 μg/mL and 40 μg/mL, respectively) alone; 3) combination (radiation and NM); and 4) vehicle (control). The same experiments were performed in vivo using a xenograft GBC mouse model. In vitro, NM inhibited radiation-induced NF-κB activity. Combination treatment significantly attenuated cell viability and increased cell apoptosis and G2/M phase cell cycle arrest compared with those in the other groups for NOZ and OCUG-1 cells. Moreover, combination treatment upregulated the expression of apoptotic proteins compared with that after the other treatments. In vivo, NM improved the antitumor action of radiation and increased the population of Ki-67-positive cells. Overall, NM enhanced the antitumor action of radiotherapy on GBC cells by suppressing radiation-induced NF-κB activity. Thus, the combination of radiotherapy and NM may be useful for the treatment of locally advanced unresectable GBC.

Efeito de inibidores de proteases na atividade enzimática de Coccus viridis em Coffea arabica

A cultura do café no Brasil tem muita importância econômica e relevância no setor social. É possível que as injurias causadas pelo inseto Coccus viridis (cochonilha-verde) seja um dos motivos da redução de sua produtividade. As plantas, ao serem injuriadas, são capazes de aumentar a síntese de inibidores de proteases (IPs) por toda sua extensão. Estudos demonstraram que insetos alimentados com plantas previamente pulverizadas com inibidores sintéticos têm seu desenvolvimento afetado. Assim, este trabalho teve por objetivo avaliar os efeitos dos inibidores de proteases sintéticos, benzamidina e berenil, sobre a atividade proteolítica intestinal de C. viridis. Plantas de Coffea arabica foram infestadas com ninfas de C. viridis e pulverizadas com benzamidina e berenil, em quatro diferentes concentrações: 0; 0,25%; 0,5% e 0,75% (p/v). A resposta bioquímica do inseto foi avaliada após 24 horas da pulverização, coletando-se os insetos. A atividade das serino-proteases tripsina-like (amidásica e esterásica) e quimotripsina-like esterásica foi reduzida pela presença dos inibidores. O mesmo se obteve em relação à atividade das cisteíno-proteases.

Effects of class-specific, synthetic, and natural proteinase inhibitors on life-history traits of the cotton bollworm Helicoverpa armigera.

Herbivorous insects have more difficulty obtaining proteins from their food than do predators and parasites. The scarcity of proteins in their diet requires herbivores to feed voraciously, thus heavily damaging their host plants. Plants respond to herbivory by producing defense compounds, which reduce insect growth, retard development, and increase mortality. Herbivores use both pre- and postdigestive response mechanisms to detect and avoid plant defense compounds. Proteinase inhibitors (PIs) are one example of plant compounds produced as a direct defense against herbivory. Many insects can adapt to PIs when these are incorporated into artificial diets. However, little is known about the effect of PIs on diet choice and feeding behavior. We monitored the diet choice, life-history traits, and gut proteinase activity of Helicoverpa armigera larvae using diets supplemented with synthetic and natural PIs. In choice experiments, both neonates and fourth-instar larvae preferred the control diet over PI-supplemented diets, to varying degrees. Larvae that fed on PI-supplemented diets weighed less than those that fed on the control diet and produced smaller pupae. Trypsin-specific PIs had a stronger effect on mean larval weight than did other PIs. A reduction of trypsin activity but not of chymotrypsin activity was observed in larvae fed on PI-supplemented diets. Therefore, behavioral avoidance of feeding on plant parts high in PIs could be an adaptation to minimize the impact of this plant's defensive strategy.

Supportive care for hemostatic complications associated with pediatric leukemia: a national survey in Japan.

Optimal supportive care for disseminated intravascular coagulation (DIC) and hemostatic complications by asparaginase is indispensable for the successful treatment of pediatric leukemia. However, the situation regarding this type of care in Japan is unclear. We conducted a questionnaire-based survey at 155 institutions treating childhood leukemia in Japan. The questionnaire asked about the supportive care provided by each institution to acute leukemia patients with DIC and asparaginase-induced hemostatic alterations. Ninety-eight institutions responded. The most common diagnostic criteria for DIC were those established by the Japanese Ministry of Health and Welfare. Regardless of the etiology underlying DIC, recombinant human thrombomodulin and synthetic protease inhibitors were used as anticoagulation therapy by around 70% and 40% of institutions, respectively. Additionally, 92%, 93%, and 73% of institutions measured plasma antithrombin, fibrinogen, and D-dimer/fibrin degradation products, respectively, more than twice per week during induction therapy for acute lymphoblastic leukemia. Survey responses indicate that 95% and 24% of the institutions used antithrombin replacement and fresh-frozen plasma, respectively. Supportive care for DIC and/or asparaginase-induced hemostatic alterations at Japanese pediatric centers was intensive and differs markedly from protocols in other countries. The efficacy of supportive care should be evaluated prospectively in the setting of pediatric leukemia.

Comparison of Danaparoid Sodium and Synthetic Protease Inhibitors for the Treatment of Disseminated Intravascular Coagulation Associated with Hematological Malignancies: A Retrospective Analysis

Background: Danaparoid sodium and synthetic protease inhibitors (SPIs) have been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan. Objectives: To compare the clinical results of the treatment of DIC with danaparoid or SPIs. Methods: We retrospectively examined 188 patients with hematological malignancy-related DIC. Results: DIC resolution rate in the danaparoid group was higher than that in the SPIs group (61.5 vs. 42.6%; p = 0.031) on day 7. Multivariate analysis identified the response to chemotherapy as independent predictive factor for DIC resolution on day 7 (odds ratio, OR, 2.28; 95% confidence interval, CI, 1.21–4.31; p = 0.011). While there was no significant difference in the DIC resolution rate on day 14 (75.0 vs. 62.4%; p = 0.117), in a subgroup analysis of patients who did not show an improvement in the underlying disease, the danaparoid group showed a significantly better DIC resolution rate (OR 3.89; 95% CI 1.15–13.2; p = 0.030). There was no difference in the rate of cumulative mortality from bleeding within 28 days between the 2 groups (6.6 vs. 3.3%; p = 0.278). Conclusions: Danaparoid may be associated with more frequent resolution of DIC in patients with refractory underlying disease.

Protease inhibitory, insecticidal and deterrent effects of the trypsin-inhibitor benzamidine on the velvetbean caterpillar in soybean.

The recognition of protease inhibitors with insecticidal activity is important as a basis for the development of mimetic peptides with potential use as biorational insecticides. We sprayed benzamidine on soybean plants and assessed whether this potent synthetic trypsin-inhibitor has protease inhibitory, insecticidal and deterrent effects on the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). Activity of trypsin inhibition in soybean leaves was increased and total proteolytic activity in the midgut extract from larvae fed on these leaves was reduced by benzamidine. Different concentrations of benzamidine sprayed on the plant caused approximately 50 % of larval mortality, and larval choice and moth preference and oviposition were all negatively affected. Low concentrations of benzamidine increased mortality and hindered insect choice and oviposition as well as higher doses. Since many synthetic protease inhibitors are usually expensive, small doses of benzamidine may be effective to protect soybean against A. gemmatalis attack. Our results highlight the potential of synthetic protease inhibitors for insecticidal and deterrent purposes in insect pest management.

Thrombomodulin alfa attenuates the procoagulant effect and cytotoxicity of extracellular histones through the promotion of protein C activation

IntroductionExtracellular histones are reported to increase thrombin generation in the plasma and induce endothelial cell death in vitro. These effects of histones were suggested to involve histone-induced inhibition of TM-dependent activated protein C (APC) generation. Therefore, we hypothesized that TM alfa, a recombinant human soluble TM, attenuates these effects of histones by promoting the generation of APC. In the present study, we investigated the effects of TM alfa on the histone-induced decrease in APC generation, an increase in thrombin generation, and endothelial cell death in vitro. MethodsAPC generation was investigated using a chromogenic substrate based assay. Thrombin generation in plasma was studied by using a calibrated automated thrombogram method. Histone cleavage was detected by western blot analysis. Histone-induced endothelial cell death was evaluated by the trypan blue exclusion test. ResultsHistones decreased APC generation and increased thrombin generation in the presence of endothelial cells. TM alfa increased APC generation and decreased thrombin generation in the presence of histones and endothelial cells. TM alfa with thrombin and protein C cleaved histone H3, and attenuated histone-induced endothelial cell death. Antithrombin, an endogenous thrombin inhibitor, and gabexate mesilate, a synthetic protease inhibitor, inhibited thrombin generation, decreased APC generation, and did not have any effect on histone H3 cleavage or histone-induced endothelial cell death. ConclusionsTM alfa attenuated the histone-induced increase in thrombin generation and endothelial cell death by promoting APC generation in vitro.

Biochemical and functional characterization of the human tissue kallikrein 9.

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+ KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

Effects of a synthetic serine protease inhibitor, camostat mesilate (FOY-305), on markers of pancreatic acinar cell damage, inflammation, and fibrosis in dogs with suspected naturally occuring chronic pancreatitis

Chronic pancreatitis is an inflammatory condition of the pancreas characterized by atrophy, fibrosis, and loss of function. Due to a lack of designated pharmaceutical agents, treatment of canine chronic pancreatitis is usually limited to supportive care. There are reports from Japan about the use of a protease inhibitor, camostat mesilate, for the treatment of chronic pancreatitis in humans, but no controlled studies about the efficacy of camostat mesilate for the treatment of chronic pancreatitis in dogs are available. Therefore, the primary goals of this study were to investigate the efficacy of camostat mesilate in dogs with suspected chronic pancreatitis as well as assess its effect on acinar cell damage by measuring serum concentrations of canine pancreas lipase immunoreactivity (cPLI). Also, the effect of the compound on systemic inflammation was quantified by measurement of serum concentrations of canine C-reactive protein and S100A12. Finally, the effect of camostat mesilate on fibrosis was assessed by measurement of serum concentrations of transforming growth factor (TGF)-β1. Thirty-one dogs with suspected chronic pancreatitis based on clinical signs and repeated measurements of an increased cPLI concentration above the cutoff value for the diagnosis of pancreatitis (≥ 400 μg/L) received a dose of 4 or 8mg/kg camostat mesilate q 8 h (12 or 24 mg/kg q 24 h) over a period of 26 ± 5 days. The evaluation of follow-up questionnaires from referring veterinarians and owners suggested improvement in quality of life in some patients. Only mild side effects were observed. Dogs given a daily camostat mesilate dose of 24 mg/kg/d orally showed a statistically significant decrease in serum cPLI concentrations between pre-treatment (median: 847 μg/L; range: 414 to 2,024 μg/L) and post-treatment (median: 520; range: 193 to 2,580 μg/L; n=19; p=0.016), indicating attenuation of acinar cell damage. Anti-inflammatory effects were comprised of a significant decrease of serum S100A12 concentrations in dogs treated with 12 mg/kg/d camostat mesilate between pre-treatment (median: 160 μg/L; range: 52 to 1327 μg/L) and post-treatment (median: 88 μg/L; range: 43 to 449 μg/L; n=11; p=0.0010). Serum cobalamin concentrations differed significantly between pre-treatment (mean: 868 ng/L; SD: ± 108ng/L) and post-treatment (mean: 695 ng/L; SD: ± 223 ng/L) concentrations in the 12 mg/kg/d group (n=12; p=0.0363) and also between pre-treatment (mean: 749 ng/L; SD: ± 229 ng/L) and post-treatment (mean: 641 ng/L; SD: ± 252 ng/L) concentrations in the 24 mg/kg/d group (n=19; p=0.045). However, no statistically significant changes in TGF-β concentrations, C-reactive protein, parameters of serum biochemistry, or complete blood counts were observed with treatment. This study would suggest a potentially beneficial role of camostat mesilate in dogs with chronic pancreatitis; however, case-control studies are needed to confirm these findings.%%%%Charakteristische Merkmale der chronischen Bauchspeicheldrusenentzundung sind Atrophie, Fibrose und…

Retraction Note to: Nafamostat mesilate inhibits the expression of HMGB1 in lipopolysaccharide-induced acute lung injury.

Purpose High mobility group box 1 (HMGB1) protein has recently been shown to be an important late mediator of acute lung injury and a promising therapeutic target. Nafamostat mesilate (NM) is a broad-range synthetic protease inhibitor with some anti-inflammatory action. The purpose of this study was to evaluate the effect of NM on HMGB1 in lipopolysaccharide (LPS)-induced lung injury in rats.

Rational design of antibody protease inhibitors.

The bovine antibody BLV1H12, which has an ultralong CDR3H, provides a novel scaffold for engineering new functions into the antibody's variable region. By modifying the β-strand "stalk" of BLV1H12 with sequences derived from natural or synthetic protease inhibitors, we have generated antibodies that inhibit bovine trypsin and human neutrophil elastase (HNE) with low nanomolar affinities. We were also able to generate a humanized variant using a human immunoglobulin scaffold that shares a high degree of homology with BLV1H12. Further optimization yielded a highly selective humanized anti-HNE antibody with sub-nanomolar affinity. This work demonstrates a novel strategy for generating antibodies with potent and selective inhibitory activities against extracellular proteases involved in human disease.

Alveolar rhabdomyosarcoma with massive disseminated intravascular coagulopathy treated with systemic chemotherapy.

It is uncommon for pediatric patients with rhabdomyosarcoma to present with clinical and/or laboratory features of disseminated intravascular coagulation (DIC). We report a case of metastatic alveolar rhabdomyosarcoma with severe bleeding because of DIC in a 13-year-old boy. He experienced persistent oozing at the site of a previous operation, gross hematuria, and massive epistaxis. Two weeks after initiating combination chemotherapy consisting of vincristine, doxorubicin, and cyclophosphamide, the patients' laboratory indications of DIC began to resolve. During this period, the patient received massive blood transfusion of a total of 311 units (26 units of red blood cells, 26 units of fresh frozen plasma, 74 units of platelet concentrates, 17 units of single donor platelets, and 168 units of cryoprecipitate), antithrombin-III and a synthetic protease inhibitor. Despite chemotherapy and radiation therapy, he died 1 year later because of disease progression. In children with metastatic rhabdomyosarcoma and massive DIC, prompt chemotherapy and aggressive supportive care is important to decrease malignancy-triggered procoagulant activities.

Responses of proteolytic enzymes in embryonic axes of germinating bean seeds under copper stress.

The changes in protease activities in embryonic axes during the first days of bean (Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos.