Cysteine Protease Inhibitors Block Toxoplasma gondii Microneme Secretion and Cell Invasion

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.

Download Full-text

Cysteine Protease Inhibitors Cure an Experimental Trypanosoma cruzi Infection

Journal of Experimental Medicine ◽

10.1084/jem.188.4.725 ◽

1998 ◽

Vol 188 (4) ◽

pp. 725-734 ◽

Cited By ~ 264

Author(s):

Juan C. Engel ◽

Patricia S. Doyle ◽

Ivy Hsieh ◽

James H. McKerrow

Keyword(s):

Trypanosoma Cruzi ◽

Protease Inhibitors ◽

Chagas Disease ◽

Golgi Complex ◽

Cysteine Protease ◽

Parasitic Infection ◽

Host Cells ◽

Vinyl Sulfone ◽

Cysteine Protease Inhibitors ◽

Lethal 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.

Download Full-text

A Toxoplasma gondii Ortholog of Plasmodium GAMA Contributes to Parasite Attachment and Cell Invasion

mSphere ◽

10.1128/msphere.00012-16 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 10

Author(s):

My-Hang Huynh ◽

Vern B. Carruthers

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Genetic Evidence ◽

Specific Role ◽

Host Cells ◽

Human Pathogen ◽

Content Type ◽

Apicomplexan Parasites ◽

Adhesive Proteins

ABSTRACT Toxoplasma gondii is a successful human pathogen in the same phylum as malaria-causing Plasmodium parasites. Invasion of a host cell is an essential process that begins with secretion of adhesive proteins onto the parasite surface for attachment and subsequent penetration of the host cell. Conserved invasion proteins likely play roles that were maintained through the divergence of these parasites. Here, we identify a new conserved invasion protein called glycosylphosphatidylinositol-anchored micronemal antigen (GAMA). Tachyzoites lacking TgGAMA were partially impaired in parasite attachment and invasion of host cells, yielding the first genetic evidence of a specific role in parasite entry into host cells. These findings widen our appreciation of the repertoire of conserved proteins that apicomplexan parasites employ for cell invasion. Toxoplasma gondii and its Plasmodium kin share a well-conserved invasion process, including sequential secretion of adhesive molecules for host cell attachment and invasion. However, only a few orthologs have been shown to be important for efficient invasion by both genera. Bioinformatic screening to uncover potential new players in invasion identified a previously unrecognized T. gondii ortholog of Plasmodium glycosylphosphatidylinositol-anchored micronemal antigen (TgGAMA). We show that TgGAMA localizes to the micronemes and is processed into several proteolytic products within the parasite prior to secretion onto the parasite surface during invasion. TgGAMA from parasite lysate bound to several different host cell types in vitro, suggesting a role in parasite attachment. Consistent with this function, tetracycline-regulatable TgGAMA and TgGAMA knockout strains showed significant reductions in host cell invasion at the attachment step, with no defects in any of the other stages of the parasite lytic cycle. Together, the results of this work reveal a new conserved component of the adhesive repertoire of apicomplexan parasites. IMPORTANCE Toxoplasma gondii is a successful human pathogen in the same phylum as malaria-causing Plasmodium parasites. Invasion of a host cell is an essential process that begins with secretion of adhesive proteins onto the parasite surface for attachment and subsequent penetration of the host cell. Conserved invasion proteins likely play roles that were maintained through the divergence of these parasites. Here, we identify a new conserved invasion protein called glycosylphosphatidylinositol-anchored micronemal antigen (GAMA). Tachyzoites lacking TgGAMA were partially impaired in parasite attachment and invasion of host cells, yielding the first genetic evidence of a specific role in parasite entry into host cells. These findings widen our appreciation of the repertoire of conserved proteins that apicomplexan parasites employ for cell invasion.

Download Full-text

Microneme Rhomboid Protease TgROM1 Is Required for Efficient Intracellular Growth of Toxoplasma gondii

Eukaryotic Cell ◽

10.1128/ec.00331-07 ◽

2008 ◽

Vol 7 (4) ◽

pp. 664-674 ◽

Cited By ~ 37

Author(s):

Fabien Brossier ◽

G. Lucas Starnes ◽

Wandy L. Beatty ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Cell Invasion ◽

Secretory Pathway ◽

Transmembrane Domain ◽

Critical Role ◽

Host Cells ◽

Wild Type ◽

Intracellular Growth ◽

Adhesive Proteins

ABSTRACT Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type T. gondii. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Serine Protease Inhibitors Block Invasion of Host Cells by Toxoplasma gondii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.6.1358 ◽

1999 ◽

Vol 43 (6) ◽

pp. 1358-1361 ◽

Cited By ~ 65

Author(s):

V. Conseil ◽

M. Soête ◽

J. F. Dubremetz

Keyword(s):

Toxoplasma Gondii ◽

Serine Protease ◽

Protease Inhibitors ◽

Early Stage ◽

Protozoan Parasite ◽

Gliding Motility ◽

Host Cells ◽

Dependent Manner ◽

Serine Protease Inhibitors ◽

Experimental Conditions

ABSTRACT We investigated the effect of protease inhibitors on the asexual development of the protozoan parasite Toxoplasma gondii. Among the inhibitors tested only two irreversible serine protease inhibitors, 3,4-dichloroisocoumarin and 4-(2-aminoethyl)-benzenesulfonyl fluoride, clearly prevented invasion of the host cells by specifically affecting parasite targets in a dose-dependent manner, with 50% inhibitory concentrations between 1 and 5 and 50 and 100 μM, respectively. Neither compound significantly affected parasite morphology, basic metabolism, or gliding motility within the range of the experimental conditions in which inhibition of invasion was demonstrated. No partial invasion was observed, meaning that inhibition occurred at an early stage of the interaction. These results suggest that at least one serine protease of the parasite is involved in the invasive process of T. gondii.

Download Full-text