Identification of Novel Malarial Cysteine Protease Inhibitors Using Structure-Based Virtual Screening of a Focused Cysteine Protease Inhibitor Library

2011 ◽  
Vol 51 (4) ◽  
pp. 852-864 ◽  
Author(s):  
Falgun Shah ◽  
Prasenjit Mukherjee ◽  
Jiri Gut ◽  
Jennifer Legac ◽  
Philip J. Rosenthal ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cysteine Protease Inhibitor ◽  
Cysteine Protease Inhibitors

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.

Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi

1998 ◽  
Vol 111 (5) ◽  
pp. 597-606 ◽  
Author(s):  
J.C. Engel ◽  
P.S. Doyle ◽  
J. Palmer ◽  
I. Hsieh ◽  
D.F. Bainton ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Golgi Complex ◽  
Cysteine Protease ◽  
Intracellular Localization ◽  
Fluorescent Microscopy ◽  
Brefeldin A ◽  
Cysteine Protease Inhibitor ◽  
Cysteine Protease Inhibitors

Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.

scholarly journals Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Bui T. T. Nga ◽  
Yuki Takeshita ◽  
Misa Yamamoto ◽  
Yoshimi Yamamoto
Keyword(s):  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Residue ◽  
Cysteine Protease Inhibitor ◽  
Inhibition Mechanism ◽  
Cysteine Protease Inhibitors ◽  
Inhibitory Potency ◽  
Inhibitory Mechanisms

Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed.

scholarly journals The complex world of plant protease inhibitors: Insights into a Kunitz-type cysteine protease inhibitor of Arabidopsis thaliana

2017 ◽  
Vol 11 (1) ◽  
pp. e1368599 ◽  
Author(s):  
Sachin Rustgi ◽  
Edouard Boex-Fontvieille ◽  
Christiane Reinbothe ◽  
Diter von Wettstein ◽  
Steffen Reinbothe
Keyword(s):  
Arabidopsis Thaliana ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cysteine Protease Inhibitor ◽  
Complex World ◽  
Plant Protease ◽  
Kunitz Type

Identification of Novel Parasitic Cysteine Protease Inhibitors by Use of Virtual Screening. 2. The Available Chemical Directory

2006 ◽  
Vol 49 (5) ◽  
pp. 1576-1584 ◽  
Author(s):  
Prashant V. Desai ◽  
Akshay Patny ◽  
Jiri Gut ◽  
Philip J. Rosenthal ◽  
Babu Tekwani ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cysteine Protease Inhibitors

Identification of Novel Parasitic Cysteine Protease Inhibitors Using Virtual Screening. 1. The ChemBridge Database

2004 ◽  
Vol 47 (26) ◽  
pp. 6609-6615 ◽  
Author(s):  
Prashant V. Desai ◽  
Akshay Patny ◽  
Yogesh Sabnis ◽  
Babu Tekwani ◽  
Jiri Gut ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cysteine Protease Inhibitors

scholarly journals Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum

1995 ◽  
Vol 305 (2) ◽  
pp. 549-556 ◽  
Author(s):  
J D Lonsdale-Eccles ◽  
G W N Mpimbaza ◽  
Z R M Nkhungulu ◽  
J Olobo ◽  
L Smith ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Leishmania Donovani ◽  
Leishmania Major ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Cysteine Protease Inhibitors ◽  
African Trypanosomes

African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.

The staphostatin family of cysteine protease inhibitors in the genus Staphylococcus as an example of parallel evolution of protease and inhibitor specificity

2007 ◽  
Vol 388 (2) ◽  
pp. 227-235 ◽  
Author(s):  
Grzegorz Dubin ◽  
Benedykt Wladyka ◽  
Justyna Stec-Niemczyk ◽  
Dorota Chmiel ◽  
Michal Zdzalik ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Parallel Evolution ◽  
Single Event ◽  
Cysteine Protease Inhibitors ◽  
Primary Target ◽  
Reciprocal Interactions ◽  
Cytoplasmic Proteins ◽  
Unique Mechanism

AbstractStaphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipocalin-like fold and a unique mechanism of action. Each of these cytoplasmic proteins is co-expressed from one operon, together with a corresponding target extracellular cysteine protease (staphopain). To cast more light on staphostatin/staphopain interaction and the evolution of the encoding operons, we have cloned and characterized a staphopain (StpA2aurCH-91) and its inhibitor (StpinA2aurCH-91) from a novel staphylococcal thiol protease operon (stpAB2CH-91) identified inS.aureusstrain CH-91. Furthermore, we have expressed a staphostatin fromStaphylococcus warneri(StpinBwar) and characterized its target protease (StpBwar). Analysis of the reciprocal interactions among novel and previously described members of the staphostatin and staphopain families demonstrates that the co-transcribed protease is the primary target for each staphostatin. Nevertheless, the inhibitor derived from one species ofStaphylococcuscan inhibit the staphopain from another species, although theKivalues are generally higher and inhibition only occurs if both proteins belong to the same subgroup of eitherS. aureusstaphopain A/staphostatin A (α group) or staphopain B/staphostatin B (β group) orthologs. This indicates that both subgroups arose in a single event of ancestral allelic duplication, followed by parallel evolution of the protease/inhibitor pairs. The tight coevolution is likely the result of the known deleterious effects of uncontrolled staphopain action.

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