A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

2020 ◽  
Author(s):  
Drake M. Mellott ◽  
Chien-Te Tseng ◽  
Aleksandra Drelich ◽  
Pavla Fajtová ◽  
Bala C. Chenna ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Host Cell ◽  
Cysteine Protease ◽  
Cleavage Site ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Protein Processing ◽  
Spike Protein ◽  
Cysteine Protease Inhibitor ◽  
Viral Infectivity

ABSTRACTK777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

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 A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 388
Author(s):  
Hương Giang Lê ◽  
A-Jeong Ham ◽  
Jung-Mi Kang ◽  
Tuấn Cường Võ ◽  
Haung Naw ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Expression Profiles ◽  
Critical Role ◽  
Cyst Formation ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Naegleria Fowleri ◽  
Nægleria Fowleri ◽  
Cystatin Family

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi

2001 ◽  
Vol 114 (21) ◽  
pp. 3933-3942 ◽  
Author(s):  
Ana C. S. Monteiro ◽  
Magnus Abrahamson ◽  
Ana P. C. A. Lima ◽  
Marcos A. Vannier-Santos ◽  
Julio Scharfstein
Keyword(s):  
Cell Surface ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Tight Binding ◽  
Cysteine Proteases ◽  
Host Cells ◽  
Cysteine Protease Inhibitor ◽  
Reversible Inhibitor ◽  
Mammalian Host

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

scholarly journals Interaction of cysteine protease inhibitor mutants with cysteine proteases

2010 ◽  
Vol 76 (2) ◽  
pp. 406
Author(s):  
S.G. Van Wyk ◽  
K.J. Kunert ◽  
B.J. Vorster ◽  
U. Schluter
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor

scholarly journals Presence of Anti-cystatin C–positive Dendritic Cells or Macrophages and Localization of Cysteine Proteases in the Apical Bud of the Enamel Organ in the Rat Incisor

2005 ◽  
Vol 53 (5) ◽  
pp. 643-651 ◽  
Author(s):  
Sumio Nishikawa
Keyword(s):  
Dendritic Cells ◽  
Cystatin C ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Enamel Organ ◽  
Apical Bud ◽  
Apical Buds ◽  
Proliferation And Differentiation

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C–positive. Anti-cystatin C–labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin ∗∗∗L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C–positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.

scholarly journals SerpinB3 drives cancer stem cell survival in glioblastoma

2021 ◽  
Author(s):  
Adam Lauko ◽  
Josephine Volovetz ◽  
Soumya M Turaga ◽  
Defne Bayik ◽  
Dennis C Watson ◽  
...  
Keyword(s):  
Cell Death ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Standard Of Care ◽  
Surface Expression ◽  
Therapeutic Interventions ◽  
Cysteine Protease Inhibitor ◽  
Cell Surface Expression ◽  
Downstream Signaling

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC therapeutic resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. The knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. Mechanistically, SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown dramatically increased the efficacy of radiation in pre-clinical models. 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 improve the efficacy of standard-of-care GBM therapies against therapeutically resistant CSCs.

scholarly journals Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

2006 ◽  
Vol 51 (3) ◽  
pp. 1064-1070 ◽  
Author(s):  
S. Eksi ◽  
B. Czesny ◽  
G.-J. van Gemert ◽  
R. W. Sauerwein ◽  
W. Eling ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Cell ◽  
Protease Inhibitor ◽  
Red Blood Cell ◽  
Cysteine Protease ◽  
Malaria Parasite ◽  
Cysteine Proteases ◽  
Food Vacuole ◽  
Cysteine Protease Inhibitor ◽  
Parasite Transmission

ABSTRACT During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.

scholarly journals Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S

Plants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1676
Author(s):  
Roberto Carlos Sallai ◽  
Bruno Ramos Salu ◽  
Rosemeire Aparecida Silva-Lucca ◽  
Flávio Lopes Alves ◽  
Thiago Henrique Napoleão ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Animal Feed ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Reversed Phase ◽  
Size Exclusion ◽  
Cysteine Protease Inhibitor ◽  
Araucaria Angustifolia ◽  
Tropical Regions ◽  
Defensive Role

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

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