Cysteine Protease Inhibitors Cure an Experimental Trypanosoma cruzi Infection

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone–derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.

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Cysteine Protease Inhibitors Block Toxoplasma gondii Microneme Secretion and Cell Invasion

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01059-06 ◽

2007 ◽

Vol 51 (2) ◽

pp. 679-688 ◽

Cited By ~ 37

Author(s):

Chin Fen Teo ◽

Xing Wang Zhou ◽

Matthew Bogyo ◽

Vern B. Carruthers

Keyword(s):

Toxoplasma Gondii ◽

Protease Inhibitors ◽

Cell Invasion ◽

Cysteine Protease ◽

Gliding Motility ◽

Host Cells ◽

Vinyl Sulfone ◽

Cysteine Protease Inhibitors ◽

Adhesive Proteins ◽

Phenyl Vinyl

ABSTRACT Toxoplasma gondii enters host cells via an active, self-driven process to fulfill its need for intracellular replication and survival. Successful host cell invasion is governed by sequential release of secretory proteins from three specialized organelles, including the micronemes, which contribute adhesive proteins necessary for parasite attachment and penetration. Cumulative evidence from studies of Trypanosoma species and malaria parasites has shown that cysteine protease inhibitors represent potent anti-parasitic agents capable of curing infections in vivo. In this study, we screened a series of selective cysteine protease inhibitors for their effects on T. gondii cell invasion. Two of these compounds, morpholinourea-leucyl-homophenolalaninyl-phenyl-vinyl-sulfone and N-benzoxycarbonyl-(leucyl)3-phenyl-vinyl-sulfone, impaired T. gondii invasion and gliding motility at low-micromolar concentrations. Unexpectedly, these inhibitors did not affect surface proteolysis of microneme products but instead impaired an earlier step by precluding the secretion of microneme-derived adhesins to the parasite surface. Our findings suggest that cysteine protease activity is required for microneme secretion and cell invasion by T. gondii.

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

Journal of Cell Science ◽

10.1242/jcs.111.5.597 ◽

1998 ◽

Vol 111 (5) ◽

pp. 597-606 ◽

Cited By ~ 6

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.

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Synthesis of quinoline derivatives as potential cysteine protease inhibitors

Future Medicinal Chemistry ◽

10.4155/fmc-2019-0201 ◽

2020 ◽

Author(s):

Marina MS Andrade ◽

Luan C Martins ◽

Gabriel VL Marques ◽

Carla A Silva ◽

Gilson Faria ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Protease Inhibitors ◽

Cysteine Protease ◽

Inhibitory Activity ◽

Molecular Targets ◽

Cysteine Proteases ◽

Leishmania Species ◽

Molecular Target ◽

Quinoline Derivatives ◽

Cysteine Protease Inhibitors

Aim: Cysteine proteases are important molecular targets involved in the replication, virulence and survival of parasitic organisms, including Trypanosoma and Leishmania species. Methodology & results: Analogs of the 7-chloro- N-[3-(morpholin-4-yl)propyl]quinolin-4-amine were synthesized and their inhibitory activity against the enzymes cruzain and rhodesain as well as against promastigotes forms of Leishmania species and epimastigotes forms of Trypanosoma cruzi were evaluated. Five compounds showed activity against both enzymes with IC50 values ranging from 23 to 123 μM. Among these, compounds 3 and 4 displayed leishmanicidal activity; compound 4 was the most promising with IC50 values <10 μM and no cytotoxicity for uninfected cells. Conclusion: The results obtained indicate that cysteine proteases are likely to be the molecular target of compounds 3 and 4.

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A QSAR study of peptidyl vinyl sulfone cysteine protease inhibitors using Topomer CoMFA and Molecular Docking

Letters in Drug Design & Discovery ◽

10.2174/1570180818666210114115030 ◽

2021 ◽

Vol 18 ◽

Author(s):

WU Lu-Yang ◽

MA Yang-Min ◽

LEI Shan ◽

WANG Tian-Hao ◽

FENG Yi

Keyword(s):

Molecular Docking ◽

Protease Inhibitors ◽

Cysteine Protease ◽

Active Site ◽

3D Qsar ◽

Vinyl Sulfone ◽

Cysteine Protease Inhibitors ◽

Qsar Study ◽

Topomer Comfa ◽

Topomer Search

Background: Malaria is one of the most important infectious diseases in the world. The most severe form of malaria in humans is caused by Plasmodium falciparum. Malaria is a worldwide health problem, with 214 million new cases in 2015 and 438,000 deaths, most of which in Africa. Therefore, there is an urgent need for novel, low-toxic, more specific inhibitors to find new antimalarial agents. A promising target for antimalarial drug design is falcipain-2, a cysteine protease from P. falciparum, that has received considerable attention due to its key role in the life cycle of the parasite. Methods: Three-dimensional quantitative structure-activity relationship (3D-QSAR) models of 39 peptidyl vinyl sulfone cysteine protease inhibitors was constructed using Topomer CoMFA. Topomer Search was employed to virtually screen lead-like compounds in the ZINC database. Molecular docking was employed to further explore the binding requirements between the ligands and the receptor protein which included several hydrogen bonds between peptidyl vinyl sulfone cysteine protease inhibitors and active site residues. Results: The non-cross correlation coefficient (r 2 ), the interaction validation coefficient (q2 ) and the external validation (r 2 pred) were 0.902, 0.685 and 0.763, respectively. The results showed that the model not only had good estimation stability but also good prediction capability. 22 new molecules were obtained, whose predicted activity are higher than template molecules. The results showed that the Topomer Search technology can be effectively applied to screen and design new peptidyl vinyl sulfone cysteine protease inhibitors. Molecular docking showed extensive interactions between peptidyl vinyl sulfone cysteine protease inhibitors and residues of LYS24, ASP21, LYS59 and ASP17 in the active site. Conclusion: 39 peptidyl vinyl sulfone cysteine protease inhibitors were used in the 3D-QSAR study. Topomer CoMFA 3DQSAR method was used to build the model, and the model was well predicted and statistically validated. The design of potent new inhibitors of cysteine protease can get useful insights from these results.

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Aziridine-2,3-Dicarboxylates, Peptidomimetic Cysteine Protease Inhibitors with Antileishmanial Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01430-05 ◽

2006 ◽

Vol 50 (7) ◽

pp. 2439-2447 ◽

Cited By ~ 38

Author(s):

Alicia Ponte-Sucre ◽

Radim Vicik ◽

Martina Schultheis ◽

Tanja Schirmeister ◽

Heidrun Moll

Keyword(s):

Protease Inhibitors ◽

Infection Rate ◽

Cysteine Protease ◽

Cysteine Proteases ◽

Host Cells ◽

Interleukin 12 ◽

Cysteine Protease Inhibitors ◽

Macrophage Infection ◽

Simultaneous Treatment ◽

Necrosis Factor Alpha

ABSTRACT Chemotherapy of leishmaniasis is mainly based on antimonials. However, they are extremely toxic and cause serious side effects, and there is a worldwide increasing frequency of chemoresistance to antimonials. These issues emphasize the urgent need for affordable alternative drugs against leishmaniasis. Leishmania cysteine proteases are essential for parasite growth, differentiation, pathogenicity, and virulence and are thus attractive targets for combating leishmaniasis. Herein we demonstrate that the cysteine protease inhibitors aziridine-2,3-dicarboxylates 13b and 13e impaired promastigote growth at mid-micromolar concentrations and decreased the infection rate of peritoneal macrophages at concentrations 8- to 13-fold lower than those needed to inhibit parasite replication. Simultaneous treatment of infected cells with compound 13b and gamma interferon resulted in an even further reduction of the concentration needed for a significant decrease in macrophage infection rate. Notably, treatment with the compounds alone modulated the cytokine secretion of infected macrophages, with increased levels of interleukin-12 and tumor necrosis factor alpha. Furthermore, the decreased infection rate in the presence of compound 13b correlated with increased nitric oxide production by macrophages. Importantly, at the concentrations used herein, compounds 13b and 13e were not toxic against fibroblasts, macrophages, or dendritic cells. Together, these results suggest that the aziridine-2,3-dicarboxylates 13b and 13e are potential antileishmanial lead compounds with low toxicity against host cells and selective antiparasitic effects.

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Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

10.1101/2021.06.04.447160 ◽

2021 ◽

Author(s):

Chia-Chuan D Cho ◽

Shuhua G Li ◽

Kai S Yang ◽

Tyler J Lalonde ◽

Ge Yu ◽

...

Keyword(s):

Protease Inhibitors ◽

Cysteine Protease ◽

Drug Repurposing ◽

Cysteine Proteases ◽

Host Cells ◽

Nonstructural Proteins ◽

Allosteric Site ◽

Cysteine Protease Inhibitors ◽

Ic50 Value ◽

Depth Analysis

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide translates ORF1a and ORF1ab in human host cells to form 15 functionally important, mature nonstructural proteins. One of the two enzymes, papain-like protease or PLpro, also possesses deubiquitination and deISGylation activities that suppresses host innate immune responses toward SARS-CoV-2 infection. Therefore, PLpro is a potential COVID-19 drug target. To repurpose drugs for PLpro, we experimentally screened 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLpro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit potent inhibition of PLpro at 200 uM. More comprehensive characterizations revealed 7 inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC50 value below 60 uM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC50 value below 10 uM. Among four inhibitors with an IC50 value below 10 uM, SJB2-043 is the most unique in that it doesn't fully inhibit PLpro but has an outstanding IC50 value of 0.56 uM. SJB2-043 likely binds to an allosteric site of PLpro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLpro holds promises but in-depth analysis of repurposed drugs is necessary to avoid omitting allosteric inhibitors.

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The Role of Sialic Acid-Binding Receptors (Siglecs) in the Immunomodulatory Effects ofTrypanosoma cruziSialoglycoproteins on the Protective Immunity of the Host

Scientifica ◽

10.1155/2013/965856 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Alexandre Morrot

Keyword(s):

Sialic Acid ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Cell Cycle Progression ◽

Parasitic Infection ◽

The United States ◽

Host Cells ◽

Health Concern ◽

Lectin Receptors ◽

Sialic Acid Binding

Chagas disease is caused by the protozoan parasiteTrypanosoma cruziand is an important endemic infection in Latin America. Lately, it has also become a health concern in the United States and Europe. Most of the immunomodulatory mechanisms associated with this parasitic infection have been attributed to mucin-like molecules on theT. cruzisurface. Mucins are high molecular weight glycoproteins that are involved in regulating diverse cellular activities in both normal and pathological conditions. InTrypanosoma cruziinfection, the parasite-derived mucins are the main acceptors of sialic acid and it has been suggested that they play a role in various host-parasite interactions during the course of Chagas disease. Recently, we have presented evidence that sialylation of the mucins is required for the inhibitory effects on CD4+T cells. In what follows we propose that signaling via sialic acid-binding Ig-like lectin receptors for these highly sialylated structures on host cells contributes to the arrest of cell cycle progression in the G1 phase and may allow the parasite to modulate the immune system of the host.

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