scholarly journals Targeted Disruption of Toxoplasma gondii Serine Protease Inhibitor 1 Increases Bradyzoite Cyst FormationIn Vitroand Parasite Tissue Burden in Mice

2011 ◽  
Vol 80 (3) ◽  
pp. 1156-1165 ◽  
Author(s):  
Viviana Pszenny ◽  
Paul H. Davis ◽  
Xing W. Zhou ◽  
Christopher A. Hunter ◽  
Vern B. Carruthers ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protease Inhibitor ◽  
Host Cell ◽  
Serine Protease ◽  
Parasitophorous Vacuole ◽  
Parasite Burden ◽  
Serine Protease Inhibitor ◽  
Genomic Locus ◽  
Content Type

As an intracellular protozoan parasite,Toxoplasma gondiiis likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities.T. gondiiserine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces twoTgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigateTgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles withDolichos bifloruslectin under conditions promotingin vitrodifferentiation. The differentiation phenotype can be partially complemented by eitherTgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and thisin vivophenotype is also complemented by eitherTgPI1 isoform. These results demonstrate thatTgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases.

scholarly journals Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

Blood ◽  
2011 ◽  
Vol 117 (4) ◽  
pp. 1176-1183 ◽  
Author(s):  
Najib El Haddad ◽  
Dean Heathcote ◽  
Robert Moore ◽  
Sunmi Yang ◽  
Jamil Azzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Protease Inhibitor ◽  
Cytotoxic T Cell ◽  
Wild Type

Abstract Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.

PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis

2014 ◽  
Vol 95 ◽  
pp. 149-156 ◽  
Author(s):  
Maram Morjen ◽  
Stéphane Honoré ◽  
Amine Bazaa ◽  
Zaineb Abdelkafi-Koubaa ◽  
Ameneallah Ellafi ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Snake Venom ◽  
Serine Protease Inhibitor ◽  
Kunitz Type

scholarly journals A Focused Small-Molecule Screen Identifies 14 Compounds with Distinct Effects on Toxoplasma gondii

2012 ◽  
Vol 56 (11) ◽  
pp. 5581-5590 ◽  
Author(s):  
Edwin T. Kamau ◽  
Ananth R. Srinivasan ◽  
Mark J. Brown ◽  
Matthew G. Fair ◽  
Erin J. Caraher ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Kinase Inhibitors ◽  
Severe Disease ◽  
Content Type ◽  
Cellular Target ◽  
Small Molecule Screen ◽  
Parasite Motility ◽  
Future Work

ABSTRACTToxoplasma gondiiis a globally ubiquitous pathogen that can cause severe disease in immunocompromised humans and the developing fetus. Given the proven role ofToxoplasma-secreted kinases in the interaction ofToxoplasmawith its host cell, identification of novel kinase inhibitors could precipitate the development of new anti-Toxoplasmadrugs and define new pathways important for parasite survival. We selected a small (n= 527) but diverse set of putative kinase inhibitors and screened them for effects on the growth ofToxoplasmain vitro. We identified and validated 14 noncytotoxic compounds, all of which had 50% effective concentrations in the nanomolar to micromolar range. We further characterized eight of these compounds, four inhibitors and four enhancers, by determining their effects on parasite motility, invasion, and the likely cellular target (parasite or host cell). Only two compounds had an effect on parasite motility and invasion. All the inhibitors appeared to target the parasite, and interestingly, two of the enhancers appeared to rather target the host cell, suggesting modulation of host cell pathways beneficial for parasite growth. For the four inhibitors, we also tested their efficacy in a mouse model, where one compound proved potent. Overall, these 14 compounds represent a new and diverse set of small molecules that are likely targeting distinct parasite and host cell pathways. Future work will aim to characterize their molecular targets in both the host and parasite.

In Vitro Dose Ranging Studies for Serine Protease Inhibitor, MK-4519, Against a Hepatitis C Virus (HCV) Replicon using the Bellocell System

2010 ◽  
Vol 86 (1) ◽  
pp. A30
Author(s):  
Ashley N. Brown ◽  
D.V. Singer ◽  
J.J. McSharry ◽  
R.J.O. Barnard ◽  
D.J. Hazuda ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Protease Inhibitor ◽  
Dose Ranging

scholarly journals Structural modeling and thermostability of a serine protease inhibitor belonging to the Kunitz family from the tick Rhipicephalus microplus

2020 ◽  
Author(s):  
Lívia de Moraes Bomediano Camillo ◽  
Graziele Cristina Ferreira ◽  
Adriana Feliciano Alves Duran ◽  
Flavia Ribeiro Santos da Silva ◽  
Wanius Garcia ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Disulfide Bonds ◽  
Tertiary Structure ◽  
Dissociation Constants ◽  
Serine Protease Inhibitor ◽  
Rhipicephalus Microplus ◽  
Human Neutrophil Elastase ◽  
Structure Stabilization

ABSTRACTrBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential, besides rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of BmTI-A was modeled based on the structure of its Sabellastarte magnifica homologue. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism data corroborated the secondary structure found by the homology modeling. Thermostability analysis confirmed the thermostability and the relation between the effects of the temperature in the inhibitor activity. The loss of activity observed was gradual, and, after 60 minutes of incubation at 90°C the inhibitor lost it completely.

scholarly journals Mutations Conferring Resistance to a Potent Hepatitis C Virus Serine Protease Inhibitor In Vitro

2004 ◽  
Vol 48 (6) ◽  
pp. 2260-2266 ◽  
Author(s):  
Liangjun Lu ◽  
Tami J. Pilot-Matias ◽  
Kent D. Stewart ◽  
John T. Randolph ◽  
Ron Pithawalla ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Protease Inhibitor ◽  
Single Amino Acid ◽  
Phenotypic Characterization ◽  
Protease Gene ◽  
Wild Type

ABSTRACT BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.

scholarly journals Spiroindolone That Inhibits PfATPase4 Is a Potent, Cidal Inhibitor of Toxoplasma gondii TachyzoitesIn VitroandIn Vivo

2013 ◽  
Vol 58 (3) ◽  
pp. 1789-1792 ◽  
Author(s):  
Ying Zhou ◽  
Alina Fomovska ◽  
Stephen Muench ◽  
Bo-Shiun Lai ◽  
Ernest Mui ◽  
...  
Keyword(s):  
Body Weight ◽  
Toxoplasma Gondii ◽  
Inhibitory Concentration ◽  
Parasite Burden ◽  
Content Type ◽  
Medicine Development ◽  
Lead Candidate ◽  
Oral Doses

ABSTRACTHere, we show that spiroindolone, an effective treatment for plasmodia, is also active againstToxoplasma gondiitachyzoites.In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC50], 1μM) and not toxic to human cells at ≥10μM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P= 0.002), measured 3 days after the last dose. This inhibition ofT. gondiitachyzoitesin vitroandin vivoindicates that spiroindolone is a promising lead candidate for further medicine development.

scholarly journals The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
William J. Blakely ◽  
Michael J. Holmes ◽  
Gustavo Arrizabalaga
Keyword(s):  
Acid Phosphatase ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Secretory Pathway ◽  
Parasitophorous Vacuole ◽  
Severe Disease ◽  
Immune Recognition ◽  
Phosphatase Domain ◽  
Content Type ◽  
Cell Defense

ABSTRACT During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

In vivo contribution of serine proteases to the proteolytic activation of γENaC in aldosterone-infused rats

2012 ◽  
Vol 303 (7) ◽  
pp. F939-F943 ◽  
Author(s):  
Kohei Uchimura ◽  
Yutaka Kakizoe ◽  
Tomoaki Onoue ◽  
Manabu Hayata ◽  
Jun Morinaga ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Proteases ◽  
Serine Protease Inhibitor ◽  
Proteolytic Activation ◽  
New Strategy ◽  
Salt Sensitive Hypertension ◽  
Primary Cleavage

Aldosterone plays an important role in the regulation of blood pressure by modulating the activity of the epithelial sodium channel (ENaC) that consists of α-, β-, and γ-subunits. Aldosterone induces a molecular weight shift of γENaC from 85 to 70 kDa that is necessary for the channel activation. In vitro experiments demonstrated that a dual cleavage mechanism is responsible for this shift. It has been postulated that furin executes the primary cleavage in the Golgi and that the second cleavage is provided by other serine proteases such as prostasin or plasmin at the plasma membrane. However, the in vivo contribution of serine proteases to this cleavage remains unclear. To address this issue, we administered the synthetic serine protease inhibitor camostat mesilate (CM) to aldosterone-infused rats. CM decreased the abundance of the 70-kDa form of ENaC and led to a new 75-kDa form with a concomitant increase in the urinary Na-to-K ratio. Because CM inhibits the protease activity of serine proteases such as prostasin and plasmin, but not furin, our findings strongly indicate that CM inhibited the second cleavage of γENaC and subsequently suppressed ENaC activity. The results of our current studies also suggest the possibility that the synthetic serine protease inhibitor CM might represent a new strategy for the treatment of salt-sensitive hypertension in humans.

scholarly journals TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

2020 ◽  
Author(s):  
Dorothea Bestle ◽  
Miriam Ruth Heindl ◽  
Hannah Limburg ◽  
Thuy Van Lam van ◽  
Oliver Pilgram ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protease Inhibitor ◽  
Host Cell ◽  
Serine Protease ◽  
Drug Targets ◽  
Serine Protease Inhibitor ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelial Cells

AbstractIn December 2019, a novel coronavirus named SARS-CoV-2 first reported in Wuhan, China, emerged and rapidly spread to numerous other countries globally, causing the current pandemic. SARS-CoV-2 causes acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. Currently, there is no approved antiviral drug for treating COVID-19 patients and there is an urgent need for specific antiviral therapies and vaccines.In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study we investigated which host cell proteases activate the SARS-CoV-2 S protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2’ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication.Our data demonstrate that both TMPRSS2 and furin are essential for SARS-CoV-2 activation in human airway cells and are promising drug targets for the treatment of COVID-19 either by targeting one of these proteases alone or by a combination of furin and TMPRSS2 inhibitors. Therefore, this approach has a high therapeutic potential for treatment of COVID-19.

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