scholarly journals A Cysteine Protease Inhibitor Rescues Mice from a Lethal Cryptosporidium parvum Infection

2013 ◽  
Vol 57 (12) ◽  
pp. 6063-6073 ◽  
Author(s):  
Momar Ndao ◽  
Milli Nath-Chowdhury ◽  
Mohammed Sajid ◽  
Victoria Marcus ◽  
Susan T. Mashiyama ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cryptosporidium Parvum ◽  
Cysteine Protease ◽  
Intestinal Inflammation ◽  
Cysteine Protease Inhibitor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
Mammalian Cell Lines

ABSTRACTCryptosporidiosis, caused by the protozoan parasiteCryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitorN-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibitsC. parvumgrowth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible toC. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in theC. parvumgenome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicityin vitroandin vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

scholarly journals Effect of phenyl vinyl sulphone cysteine protease inhibitor on Schistosoma mansoni: in vitro and in vivo experimental studies

2017 ◽  
Vol 41 (4) ◽  
pp. 1049-1058
Author(s):  
Manal Salah El-Din Mahmoud ◽  
Ayman Nabil Ibrahim ◽  
Abeer Fathy Badawy ◽  
Nourhan Mohamed Abdelmoniem
Keyword(s):  
Protease Inhibitor ◽  
Schistosoma Mansoni ◽  
Cysteine Protease ◽  
Experimental Studies ◽  
Cysteine Protease Inhibitor ◽  
Phenyl Vinyl

scholarly journals Interaction of Rifampin and Darunavir-Ritonavir or Darunavir-Cobicistat In Vitro

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Owain Roberts ◽  
Saye Khoo ◽  
Andrew Owen ◽  
Marco Siccardi
Keyword(s):  
High Performance ◽  
Linear Regression Analysis ◽  
Intrinsic Clearance ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
Pbpk Models ◽  
Physiologically Based Pharmacokinetic ◽  
Dosing Regimens

ABSTRACT Treatment of HIV-infected patients coinfected with Mycobacterium tuberculosis is challenging due to drug-drug interactions (DDIs) between antiretrovirals (ARVs) and antituberculosis (anti-TB) drugs. The aim of this study was to quantify the effect of cobicistat (COBI) or ritonavir (RTV) in modulating DDIs between darunavir (DRV) and rifampin (RIF) in a human hepatocyte-based in vitro model. Human primary hepatocyte cultures were incubated with RIF alone or in combination with either COBI or RTV for 3 days, followed by coincubation with DRV for 1 h. The resultant DRV concentrations were quantified by high-performance liquid chromatography with UV detection, and the apparent intrinsic clearance (CLint.app.) of DRV was calculated. Both RTV and COBI lowered the RIF-induced increases in CLint.app. in a concentration-dependent manner. Linear regression analysis showed that log10 RTV and log10 COBI concentrations were associated with the percent inhibition of RIF-induced elevations in DRV CLint.app., where β was equal to −234 (95% confidence interval [CI] = −275 to −193; P < 0.0001) and −73 (95% CI = −89 to −57; P < 0.0001), respectively. RTV was more effective in lowering 10 μM RIF-induced elevations in DRV CLint.app. (half-maximal [50%] inhibitory concentration [IC50] = 0.025 μM) than COBI (IC50 = 0.223 μM). Incubation of either RTV or COBI in combination with RIF was sufficient to overcome RIF-induced elevations in DRV CLint.app., with RTV being more potent than COBI. These data provide the first in vitro experimental insight into DDIs between RIF and COBI-boosted or RTV-boosted DRV and will be useful to inform physiologically based pharmacokinetic (PBPK) models to aid in optimizing dosing regimens for the treatment of patients coinfected with HIV and M. tuberculosis.

scholarly journals A Phytophthora palmivora Extracellular Cystatin-Like Protease Inhibitor Targets Papain to Contribute to Virulence on Papaya

2018 ◽  
Vol 31 (3) ◽  
pp. 363-373 ◽  
Author(s):  
Rebecca Gumtow ◽  
Dongliang Wu ◽  
Janice Uchida ◽  
Miaoying Tian
Keyword(s):  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Gene Editing ◽  
Sequence Data ◽  
Cysteine Protease Inhibitor ◽  
Phytophthora Palmivora ◽  
Cysteine Protease Inhibitors ◽  
Successful Infection

Papaya fruits, stems, and leaves are rich in papain, a cysteine protease that has been shown to mediate plant defense against pathogens and insects. Yet the oomycete Phytophthora palmivora is a destructive pathogen that infects all parts of papaya plants, suggesting that it has evolved cysteine protease inhibitors to inhibit papain to enable successful infection. Out of five putative extracellular cystatin-like cysteine protease inhibitors (PpalEPICs) from P. palmivora transcriptomic sequence data, PpalEPIC8 appeared to be unique to P. palmivora and was highly induced during infection of papaya. Purified recombinant PpalEPIC8 strongly inhibited papain enzyme activity, suggesting that it is a functional cysteine protease inhibitor. Homozygous PpalEPIC8 mutants were generated using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation (AMT). Increased papain sensitivity of in-vitro growth and reduced pathogenicity during infection of papaya fruits were observed for the mutants compared with the wild-type strain, suggesting that PpalEPIC8, indeed, plays a role in P. palmivora virulence by inhibiting papain. This study provided genetic evidence demonstrating that plant-pathogenic oomycetes secrete cystatins as important weapons to invade plants. It also established an effective gene-editing system for P. palmivora by the combined use of CRISPR/Cas9 and AMT, which is expected to be applicable to other oomycetes.

scholarly journals Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba

2013 ◽  
Vol 12 (4) ◽  
pp. 567-574 ◽  
Author(s):  
Jung-Yub Lee ◽  
Su-Min Song ◽  
Eun-Kyung Moon ◽  
Yu-Ran Lee ◽  
Bijay Kumar Jha ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Protease Activity ◽  
Cathepsin L ◽  
Cyst Formation ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Content Type ◽  
Human Cathepsin ◽  
Interfering Rna

ABSTRACTThe encystation ofAcanthamoebaleads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protectAcanthamoebafrom intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established inAcanthamoeba. In the present study, we identified and characterizedAcanthamoebacysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation ofAcanthamoeba.

scholarly journals In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

2012 ◽  
Vol 53 (1) ◽  
pp. 100-105 ◽  
Author(s):  
Milica Popovic ◽  
Uros Andjelkovic ◽  
Milica Grozdanovic ◽  
Ivana Aleksic ◽  
Marija Gavrovic-Jankulovic
Keyword(s):  
Antibacterial Activity ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Actinidia Deliciosa ◽  
Cysteine Protease Inhibitor ◽  
In Vitro Antibacterial Activity

scholarly journals Discovery of a AhR flavonoid agonist that counter-regulates ACE2 expression in rodent models of inflammation and attenuates ACE2-SARS-CoV2 interaction in vitro

2021 ◽  
Author(s):  
Michele Biagioli ◽  
Silvia Marchianò ◽  
Rosalinda Roselli ◽  
Cristina Di Giorgio ◽  
Rachele Bellini ◽  
...  
Keyword(s):  
Viral Entry ◽  
Intestinal Inflammation ◽  
Binding Pocket ◽  
Docking Studies ◽  
Spike Protein ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Fatty Acid Binding

AbstractThe severe acute respiratory syndrome (SARS)-CoV-2, a newly emerged coronavirus first identified in 2019, is the pathogenetic agent od Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE) 2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of ACE2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin, a natural flavonoid bind and activates the Aryl hydrocarbon Receptor (AhR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent DSS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of ACE2 mRNA expression. Colon levels of ACE2 mRNA were directly correlated with TNFα mRNA levels. In contrast to ACE2 the angiotensin 1-7 receptor MAS was downregulated in the inflamed tissues. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV2 Spike protein to ACE2 and reduces the SARS-CoV2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.

STEM-15. SerpinB3 DRIVES CANCER STEM CELL SURVIVAL IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi24-vi24
Author(s):  
Adam Lauko ◽  
Soumya M Turaga ◽  
Josephine Volovetz ◽  
Defne Bayik ◽  
Shideng Bao ◽  
...  
Keyword(s):  
Cell Death ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Xenograft Model ◽  
Standard Of Care ◽  
Therapeutic Interventions ◽  
Cysteine Protease Inhibitor ◽  
Orthotopic Xenograft Model

Abstract Despite therapeutic interventions for glioblastoma (GBM), self-renewing, therapy-resistant populations of cells referred to as cancer stem cells (CSCs) drive recurrence. Previously, we identified the unique expression of junctional adhesion molecule-A (JAM-A) on CSCs and demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. Moreover, we determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the entire signaling network has yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from patient-derived GBM CSCs and performed mass spectrometry to determine JAM-A binding proteins. This led to the identification of the cysteine protease inhibitor SerpinB3 as a putative JAM-A binding partner. Using in vitro CSC functional assays, we show that SerpinB3 is necessary for CSC maintenance and survival. In an in vivo orthotopic xenograft model, knockdown of SerpinB3 extended survival. Mechanistically, knockdown of SerpinB3 led to decreased expression of TGF-β, Myc, WNT, and Notch signaling, known regulators of the CSC state. Additionally, knockdown of SerpinB3 increases susceptibility to radiation therapy. SerpinB3 is essential for buffering cells against cathepsin-mediated cell death, and we found that elevated lysosomal membrane permeability after radiation leads to cathepsin release into the cytoplasm. As a result, SerpinB3 knockdown cells have a diminished capacity to inhibit cathepsin-driven cell death after radiation. The addition of the cathepsin inhibitor E64D partially rescues the SerpinB3 knockdown, however, SerpinB3 mutants that are unable to inhibit cathepsins fail to do the same. Taken together, our findings, identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to increase the efficacy of standard of care GBM therapies against therapy-resistant CSCs.

scholarly journals In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

2014 ◽  
Vol 56 (1) ◽  
pp. 43-47 ◽  
Author(s):  
Thais Batista de Carvalho ◽  
Teresa Cristina Goulart Oliveira-Sequeira ◽  
Semiramis Guimaraes
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Cysteine Protease Inhibitor ◽  
Parasite Survival ◽  
Dye Reduction ◽  
Antigiardial Activity ◽  
Host Parasite ◽  
Druggable Targets ◽  
Vitro Effect

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.

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